Twenty years after the first reported clinical evidence of AIDS, more than 60 million people have been infected with HIV-1. The clinical course and outcome of HIV-1 infection are characterized by extreme heterogeneity among untreated infected individuals, with the majority of individuals progressing to AIDS during an average time of 10 years. In contrast, a small subset of infected individuals maintains low to undetectable HIV-1 viral loads, stable and normal CD4 T-cell counts and has no manifestations of HIV-1 disease despite documented infection with HIV-1, in some cases for more than 20 years [1–9]. These differences in the clinical course of HIV-1 infection may represent a result of genetic variants among the infecting HIV-1 strains, host genetic differences and/or differences in the virus-specific immune response [6, 10–18].
Several cohort studies examining HLA associations with disease progression in infected individuals have suggested that heterozygosity of the HLA class I loci is associated with the delayed onset of AIDS [15,19,20], and a number of HLA-B alleles, in particular HLA-B57, have been associated with low set-point viremia and long-term non-progressive chronic HIV-1 infection in multiple independent studies [12,16–26]. One possible explanation for this association is that the genetic HLA class I background influences the quality and efficiency of the immune responses generated against HIV-1. Since early events in acute HIV-1 infection have been hypothesized to influence subsequent disease progression, we examined persons with acute HIV-1 infection to determine the relative immunodominance of HLA-B57-restricted CD8 T-cell responses during the acute immune response and the subsequent contribution of HLA-B57 to chronic immune control.
Material and methods
Study subjects expressing the HLA class I allele B57 were recruited from two large ‘Acute Infection Cohorts’ at the Massachusetts General Hospital in Boston [27,28] and the San Francisco General Hospital . In addition, two individuals with early (< 180 days) HIV-1 infection expressing HLA-B57 were recruited from the Department of Medicine of the University Hospital in Bonn, Germany (study subject MS), and the Institut de Recherches Cliniques de Montreal, Canada (study subject PI004 ). HLA class I molecular typing was performed using single-strand polymorphism (SSP)–PCR . These studies were approved by the local Institutional Review Boards and all individuals gave informed consent for participation in the study.
Frozen peripheral blood mononuclear cells (PBMC) were plated on 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore, Bedford, Massachusetts, USA) that had been previously coated with 100 μl of an anti-interferon (IFN)-γ monoclonal antibody (mAB) 1-D1k (0.5 μg/ml; Mabtech, Stockholm, Sweden) overnight at 4°C. Peptides corresponding to described optimal CTL epitopes  or overlapping peptides spanning the expressed HIV-1 clade B consensus sequence  were added directly to the wells at a final concentration of 1 × 10−5 M. Cells were added to the wells at 50 000–100 000 cells per well. The plates were incubated at 37°C, 5% CO2 overnight (14–16 h) and then processed as described previously [33,34]. IFN-γ producing cells were counted by direct visualization and are expressed as spot forming cells (SFC) per 1 × 106 cells. The number of specific IFN-γ secreting T cells was calculated by subtracting the negative control value. Responses ≥ 50 SFC/1 × 106 input cells after subtraction of background activity and higher than three times mean background activity were considered positive. The positive control consisted of incubation of 100 000 PBMC with phytohaemagglutinin.
Flow cytometric detection of antigen-induced intracellular IFN-γ
Intracellular cytokine staining assays were performed as described elsewhere with minor modifications [35,36]. Briefly, 0.5 × 106−1.0 × 106 fresh PBMC were incubated with 2 μM peptide and 1 μg/ml each of the mAb anti-CD28 and anti-CD49d (Becton Dickinson, Mountain View, California, USA) at 37°C, 5% CO2, for 1 h, before the addition of 10 μg/ml Brefeldin A (Sigma, St Louis, Missouri, USA). Following a further 5-h incubation, the cells were placed at 4°C overnight. After surface-marker labeling with anti-CD8 and anti-CD4 (Becton Dickinson), cells were fixed and permeabilized using Caltag Fixation/Permeabilization Kit (Caltag, Burlinghame, California, USA) and anti-IFN-γ-mAb (Becton Dickinson) was added. Cells were analyzed on a FACSort Flowcytometer (Becton Dickinson Immunocytometry systems, San Jose, California, USA). Control conditions were established by the use of autologous PBMC, which had not been stimulated with peptide, but otherwise had been treated identically.
To determine the contribution of responses restricted by individual HLA class I alleles to the total HIV-1-specific CD8 T-cell response, autologous and partially HLA-matched Epstein–Barr virus-transformed B-lymphoblastoid cell lines (BCL) were pulsed with pools of overlapping peptide (pool A, 66 HIV-1 Gag peptides; pool B, 113 HIV-1 Env peptides; pool C, 133 HIV-1 Pol peptides; pool D, 98 HIV-1 Nef, Rev, Tat, Vpr, Vif and Vpu peptides), at a final concentration of 5 μg/ml per peptide for 1 h and then washed five times prior to incubation with effector PBMC (105 BCL and 5 × 105 PBMC) [37,38]. The mAbs anti-CD28 and anti-CD49d were subsequently added and the assay run exactly as described above.
Statistical analysis and graphical presentation was done using SigmaPlot 5.0 (SPSS Inc., Chicago, Illinois, USA). Results are given as mean ± standard deviation (SD) or median with range. Statistical analysis of significance (P values) were based on two-tailed t tests and chi-squared test.
Individuals expressing HLA-B57 present less frequently with symptomatic acute HIV-1 infection syndrome than individuals not expressing this allele
The median viral load set point following untreated acute HIV-1 infection is approximately 30 000 HIV-1 RNA copies/ml plasma at 1 year . However, rare persons with sustained low viremia following acute infection have been described, including an HIV-1-infected individual (PI004) with early HIV-1 infection who presented with a viral load of 63 copies/ml 7 weeks after the first positive HIV-1 antibody test we reported earlier . This individual expressed the HLA-B57 allele, an allele that has been associated with long-term non-progressive chronic HIV-1 infection. The observation of spontaneous control of HIV-1 viremia following acute HIV-1 infection in this individual expressing HLA-B57, together with prior data describing restriction of viral replication in HLA-B57 positive individuals with chronic infection [16–18], led us to hypothesize that HLA-B57 is associated with rapid control of viremia during acute HIV-1 infection. In order to test this hypothesis, we examined the relationship between HLA-B57 and clinical presentation in acute HIV-1 infection in two large ‘Acute HIV-1 Infection Cohorts’ established in Boston and San Francisco. Individuals with symptomatic acute HIV-1 infection (defined as an acute viral syndrome in the presence of detectable HIV-1 viremia and either a negative ELISA or a positive ELISA and less than three bands in an HIV-1 Western blot) were identified from the two cohorts. A total of 59 individuals from San Francisco and 57 individuals from Boston had presented with acute symptomatic HIV-1 infection and were typed for HLA class I alleles at the time of the study. Only four of these 116 individuals (3.4%) expressed the HLA class I allele B57. The low prevalence of HLA-B57 in individuals with symptomatic acute HIV-1 infection was significantly different from the percentage of individuals expressing this allele in the general HIV-1 infected population (9.6%; P < 0.05), as determined by HLA class I typing of 446 infected individuals from the Boston area. These differences in the frequency of HLA-B57 were not due to differences in the ethnicities of the studied cohorts, as the prevalence of non-Caucasians ranged from 20% to 22% in these cohorts (P = 0.9). The phenotypic HLA-B57 frequency of 9.6% (43/446) determined in the HIV-1-infected population in this study is in line with previous reports on the frequency of HIV-1-infected individuals expressing HLA-B57, ranging from 7% to 10.8% [12,18,40], as well as in the general USA population (11%) [18,41]. In contrast to HLA-B57, other HLA class I alleles frequently expressed in the studied population, including HLA-A2, -A3, -B7, -B8 and -B35, were expressed at similar frequencies in individuals with symptomatic acute HIV-1 infection and in the general HIV-1 infected population in Boston (P > 0.3). Taken together, the low frequency of detection of HLA-B57-expressing persons at the time of acute HIV-1 infection, coupled with the statistically higher percentage of HLA-B57 positive persons with chronic HIV-1 infection, suggests that symptomatic acute infection, and therefore opportunity to diagnose infection, is less frequent in persons who are HLA-B57 positive.
HIV-1-specific CD8 T-cell responses during acute and early HIV-1 infection are dominated by broadly directed HLA-B57-restricted responses in individuals expressing this allele
To assess the influence of HLA-B57 on cellular immune responses during acute HIV-1 infection, we identified six additional HLA-B57 positive individuals diagnosed within 6 months of infection, all of whom had experienced asymptomatic acute HIV-1 infection, including the pilot subject PI004. The clinical characteristics of all nine HLA-B57 positive individuals for whom frozen PBMC samples were available to assess HIV-1-specific T-cell responses are shown in Table 1. Of note, the majority (6/9) of individuals expressing HLA-B57 were able to control viral replication in the absence of antiretroviral therapy at levels < 5000 copies HIV-1 RNA/ml plasma (median, 515 copies/ml) during up to 29 months following acute infection.
HIV-1-specific CD8 T-cell responses have been associated with the initial control of viral replication during acute AIDS virus infection in macaques and humans [42–46]. In order to assess the contribution of HLA-B57-restricted CD8 T-cell responses to the total HIV-1-specific CD8 T-cell responses, CD8 T-cell responses were characterized in all nine HLA-B57 positive individuals, using the first PBMC sample available (3 weeks to 6 months following seroconversion; median, 4 months; Table 1) and all known optimal CTL epitopes described for their HLA class I type . The average number of peptides tested was 27 (range, 22–39). In all individuals, PBMC responded to non-specific stimulation using phytohaemagglutinin with IFN-γ production of T cells, demonstrating functional activity of the cryopreserved cells. Overall, total HIV-1-specific CD8 T-cell responses ranged from 0 SFC/1 × 106 PBMC to 5350 SFC/1 × 106 PBMC (median, 1630 SFC/1 × 106 PBMC; Fig. 1). We detected a median of six different CTL epitopes (range 0–10) targeted in these nine subjects. HLA-B57-restricted responses dominated the total HIV-1-specific responses, contributing a median of 74% to the total responses (P < 0.03; Fig. 1) and restricting a median of four different epitopes, compared to a median of one epitope restricted by all additional five major HLA class I alleles (P < 0.02; Fig. 1). We compared this relative immunodominance of HLA-B57 restricted CD8 T-cell responses during acute HIV-1 infection in individuals expressing this allele to the relative immunodominance of responses restricted by other expressed HLA class I alleles for which a comparable number of optimal CTL epitopes have been described [28,31]. These alleles included HLA-A2, -A3, -B7, and -B8, the HLA class I alleles most commonly expressed in the Caucasian population studied. For none of these alleles epitope-specific T-cell responses restricted by a single allele dominated total HIV-1-specific T-cell responses (Fig. 2). In contrast, total CD8 T-cell responses restricted by HLA-A2 were rarely detectable during acute infection, as described previously [28,29].
Of the 11 described HLA-B57-restricted CD8 T-cell epitopes tested in this study , 10 were recognized by at least one individual following HIV-1 infection (Table 2). The most frequently targeted epitopes were the p24 Gag epitopes KAFSPEVIPMF (KF11) and TSTLQEQIGW (TW10) and the reverse transcriptase (RT) epitope IVLPEKDSW (IW9) (Table 2). The epitopes IW9 in RT and TW10 in p24 Gag also represented the immunodominant HLA-B57-restricted CTL epitopes in seven out of nine individuals (Table 2). One individual (AC-34), who was treated during symptomatic acute infection prior to HIV-1 seroconversion and maintained undetectable viral load throughout the treatment period, had no detectable HIV-1-specific CD8 T-cell responses against described optimal CTL epitopes on multiple occasions studied during the 22-month follow-up period. In this person, comprehensive assessment of HIV-1-specific CD8 T-cell responses using a set of 410 overlapping peptides spanning the entire expressed HIV-1 clade B consensus sequence confirmed the presence of only very weak virus-specific responses, with responses to a total of three out of 410 overlapping peptides located within p24 Gag, RT and Nef (100, 80 and 160 SFC/1 × 106 PBMC, respectively). None of these three peptides contained epitopes described for the individual's HLA class I type. Taken together these data demonstrate that HLA-B57-restricted CD8 T-cell responses significantly dominate total HIV-1-specific CD8 T-cell responses in the majority of studied individuals and can target a large variety of different CD8 T-cell epitopes including the immunodominant virus-specific CD8 T-cell responses during this early phase of infection.
Immunodominance of HLA-B57-restricted HIV-1-specific CD8 T-cell responses persists following primary HIV-1 infection
The above data demonstrate that HLA-B57-restricted CD8 T-cell responses are dominant in primary HIV-1 infection and that individuals expressing HLA-B57 are less likely to have symptoms during acute infection. We subsequently assessed whether these responses remain the immunodominant responses later in infection. For six individuals, multiple follow-up samples over a median of 18.5 months (range, 8–29 months) after HIV-1 diagnosis were available and studied for HIV-1-specific CD8 T-cell responses using the methods described above (Fig. 3). Two of these individuals (AC-43 and AC-75) had started antiretroviral treatment following primary infection, the other four individuals remained off therapy during the study period with viral loads ranging from 123 to 4383 (median, 515) copies HIV-1 RNA/ml plasma (Table 1). In all individuals, HLA-B57-restricted CD8 T-cell responses remained the dominant HIV-1-specific responses (Fig. 3). However, responses to additional epitopes restricted by HLA-B57 and other HLA class I alleles developed over time with continuous or intermittent exposure to viral antigen and total HIV-1-specific CD8 T-cell responses increased from a median of 1325 SFC/1 × 106 PBMC to a median of 2030 SFC/1 × 106 PBMC (P = 0.1), while the contribution of HLA-B57-restricted responses to the total HIV-1-specific CD8 T-cell responses remained stable (71% versus 72% during earliest timepoint, data not shown). Overall, the significant immunodominance of HLA-B57-restricted HIV-1-specific CD8 T-cell responses persisted over time.
The use of described optimal CD8 T-cell epitopes in the assessment of HIV-1-specific CD8 T-cell responses may underestimate the total magnitude and breadth of responses, as potential responses to as yet undefined epitopes will be missed [28,32,47]. We therefore confirmed the immunodominance of HLA-B57-restricted HIV-1-specific CD8 T-cell responses in subject AC-38 for whom sufficient cells were available to undertake this type of evaluation 10 months after infection, using a comprehensive assessment of the contribution of each HLA class I allele to the total HIV-1-specific CD8 T-cell response by flow-based quantification of antigen-specific IFN-γ production. The evaluation of this technique showed that it allows for the reproducible quantification of the contribution of CD8 T-cell responses restricted by each expressed HLA class I allele to the total virus-specific CD8 T-cell responses ([37,38] and data not shown). In AC-38, a total of 5.7% of CD8 T cells responded to stimulation with autologous BCL pulsed with the four HIV-1 overlapping peptide pools (Fig. 4). This magnitude of responses to the autologous antigen-presenting cells was similar to the cumulative magnitude of responses to the overlapping peptide pools presented on partially HLA-matched antigen-presenting cells (6.1%; P = 0.92). The described optimal HLA-B57-restricted peptide KAFSPEVIPMF (KF11) Gag was used as a control peptide and was only presented by HLA-B57-expressing cell lines (Fig. 4). CD8 T-cell responses to BCL matched by HLA-B57 contributed more than 60% to the total HIV-1-specific CD8 T-cell responses, followed by HLA-A3/B7/Cw7-restricted responses (25%) and HLA-A1-matched responses (15%). This contribution of the different HLA class I alleles to the total HIV-1-specific CD8 T-cell response measured by the comprehensive intracellular cytokine staining approach corresponded well to the contribution determined at the same time point by the use of described optimal CD8 T-cell epitopes for each HLA type (HLA-B57: 60%, -A1: 5%, -A3: 34% and -B7: 3%; data not shown), and confirmed the immunodominance of HLA-B57-restricted CD8 T-cell responses.
Several studies have demonstrated the crucial role of cellular immune responses generated during acute HIV-1 or SIV infection in the initial control of viremia and the establishment of viral set point [6,27,42–46,48]. Here we extended these studies and determined the impact of the genetic HLA class I background on the magnitude, breadth and maintenance of virus-specific CD8 T-cell responses in acute HIV-1 infection and during the transition to chronic infection. The data presented in this study demonstrate that the expression of the HLA class I allele B57 is significantly associated with the absence of a symptomatic HIV-1 seroconversion illness, and that CD8 T-cell responses restricted by this allele dominate not only the initial HIV-1-specific immune response in individuals expressing this allele, but also dominate in terms of magnitude and breadth of responses during the chronic phase of infection.
Acute HIV-1 infection is a very heterogeneous syndrome and individuals presenting with more severe symptoms during acute infection and a longer duration of the acute infection syndrome tend to progress more rapidly to AIDS [49–54]. The factors influencing this association between severity of acute HIV-1 infection syndrome and disease outcome are not understood. Our data demonstrate that individuals expressing HLA-B57, an HLA class I allele associated with long-term non-progressive HIV-1 infection, present significantly less frequently with acute symptomatic HIV-1 infection (P < 0.05), suggesting a crucial impact of this allele on the course of acute infection. These studies also provide the first evidence of a link between HLA-B57 and enhanced early immune control of HIV-1 viremia, thereby extending studies in chronic infection describing an association between HLA-B57 and long-term non-progressive infection [12,16–26]. Six of the nine individuals studied never required the initiation of antiretroviral treatment and controlled HIV-1 viremia at levels < 5000 copies/ml, and at several occasions below the limit of detection (< 50 copies/ml). The three remaining study subjects presented with symptomatic acute infection and required initiation of antiretroviral therapy, indicating that additional factors other than the expression of HLA-B57 may influence clinical presentation and viral control during acute infection. However, the control of viremia in the majority of infected individuals is remarkable, and differs importantly from the viral set point normally observed in infected individuals during the first years of infection . These data suggest that the low viral set-point that ultimately predicts a slow progression to AIDS is already established early in infection in individuals expressing HLA class I alleles associated with long-term non-progressive infection.
This study also provides a functional association between HLA-B57 expression and dominant CD8 T-cell responses restricted by this allele early in infection. Overall, a median of 74% of the total magnitude of early HIV-1-specific responses were restricted by HLA-B57, and CTL epitopes restricted by this allele contributed a median of 80% to the total breadth of responses. This dominance of HLA-B57-restricted CD8 T-cell responses during primary HIV-1 infection was highly significant and persisted during up to 29 months of follow-up. Our data using a comprehensive flow-based assessment of total virus-specific CD8 T-cell response directed against all expressed HIV-1 proteins confirmed the dominance of HLA-B57- restricted responses. However, as total virus-specific CD8 T-cell responses depend on both the HLA class I type of the individual and genetic variations of the particular HIV-1 strain infecting that individual, the use of peptides based on HIV-1 consensus sequences in this study may still have underestimated total virus-specific responses. The ability of HLA-B57 to present a large variety of CD8 T-cell epitopes within different HIV-1 proteins, including a broad functional cross-reactivity to both common and rare variants of these epitopes, has been described in several studies [22–24,55]. In addition, recent data in HIV-1 negative volunteers vaccinated with an ALVAC-HIV recombinant canarypox vaccine demonstrate that individuals expressing HLA-B57 and HLA-B27, another allele associated with slower progression of natural HIV-1 infection, developed significantly more frequently CD8 T-cell responses to the vaccine than individuals not expressing these alleles . Taken together, these data suggest that the polymorphism of HLA class I alleles not only influences the history of natural HIV-1 infection, but also modulates the responses to HIV-1 vaccines. The dissection of the molecular interactions responsible for the advantages of these HLA class I alleles in presenting HIV-1 antigens will be crucial for our understanding of HLA determinants of virus-induced CD8 T-cell responses.
In conclusion, we observed an association between HLA-B57, an allele associated with slow progression in HIV-1 infection, and favorable clinical, virological and immunological events during the critical early stage of HIV-1 infection. HLA-B57-positive individuals presented significantly less frequently with symptoms during acute HIV-1 infection, and the majority of individuals expressing this allele controlled HIV-1 replication in the absence of therapy. This control of HIV-1 viremia was associated with immunodominant virus-specific CD8 T-cell responses restricted by HLA-B57. Taken together, these data suggest that during acute HIV-1 infection the interaction between the genetic HLA class I background and the viral epitopes presented may determine the efficiency of virus-specific CD8 T-cell responses, influencing viral setpoints, clinical disease and long-term disease outcome.
Sponsorship: Supported by the Doris Duke Charitable Foundation (MA, ESR and BDW), the National Institutes of Health, the Foundation for AIDS & Immune Research (MA), the American Foundation for AIDS Research (MMA), the Concerned Parents for AIDS Research (MMA), the Partners/Fenway/Shattuck Center for AIDS Research (MA and XGY) and the Howard Hughes Medical Institute (BDW). MA is a recipient of a Doris Duke Clinical Scientist Development Award, BDW is a recipient of a Doris Duke Distinguished Clinical Scientist Award and PJRG is an Elisabeth Glaser Scientist of the Pediatric AIDS Foundation.
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